This is an application for a K08 award for Dr. Rebecca Gillani, a neurologist at the Massachusetts General Hospital. She has rigorous prior training in neuroplasticity in rodent models of a neurodevelopmental disorder and stroke. In her initial post-doctoral experience, she has gained expertise in in vivo two-photon imaging technology working on a project to define basic principles of circuit wiring in the visual cortex. She now aims to take these state-of-the-art techniques for in vivo two-photon imaging of neurons and synapses and apply them to the study of neurodegeneration in rodent preclinical models of multiple sclerosis, her area of clinical interest. The goal of this career development award is to provide her the skills to make the transition from the study of physiologic circuit wiring and plasticity, to the independent study of disease models of neuroinflammation with training as follows: 1) Multi-disciplinary mentoring from experts in neurodegeneration, neuroimmunology, and in vivo two-photon imaging; 2) methodology for experimental autoimmune encephalomyelitis mouse model of multiple sclerosis; 3) didactic training in immunology and biostatistical methodology; and 4) communication and leadership career development. Her mentor Brian Bacskai is expert in in vivo two-photon imaging in neurodegenerative diseases, and her co-mentor Elly Nedivi is expert in in vivo two-photon imaging of neuron and synapse structure. Her advisors include experts in neurodegeneration, Rudolph Tanzi, and experts in neuroimmunology, Francisco Quintana and Staci Bilbo. While there is an immense literature and ongoing effort to define the immune mechanisms underlying multiple sclerosis pathology, a critical area of unmet need is defining the specific mechanisms of neuronal and synaptic dysfunction and degeneration during the course of multiple sclerosis. These neurodegenerative changes are a key determinant of disability in multiple sclerosis. It is likely that inflammation in early multiple sclerosis does not immediately lead to synapse loss. Instead there is more likely a series of evolving changes in synapse structural integrity and dynamics with the eventual outcome of synapse loss. In Aim 1, Dr. Gillani will leverage state-of-the- art in vivo two-photon imaging technologies for longitudinal monitoring of all the excitatory and inhibitory connections onto individual cortical pyramidal neurons in a living mouse in a model of multiple sclerosis. In Aim 2, Dr. Gillani will use new methods for reversible expansion and super-resolution imaging of cellular architecture to analyze for the presence of important synaptic proteins and inflammatory factors implicated in synaptic pathology in multiple sclerosis, by observing the same neurons and synapses imaged in live animals in Aim 1. The studies she proposes will provide a comprehensive picture of how diverse populations of synapses across the entire dendritic tree of cortical pyramidal cells undergo dynamic changes over 8 weeks in a mouse model of multiple sclerosis. This research will be the basis of a R01 application in year 4 of this proposed K08 award.